Polyamide nucleic acid derivatives and agents, and processes for preparing them

ABSTRACT

The present invention relates to PNA derivatives which carry, at the C terminus, or at both the C and N termini of the PNA backbone, one or more phosphoryl radicals. The phosphoryl radicals carry, where appropriate, one or more labeling groups, groups for crosslinking, groups which promote intracellular uptake, or groups which increase the binding affinity of the PNA derivative for nucleic acids. The invention furthermore relates to a process for preparing the above-mentioned PNA derivatives and to their use as pharmaceuticals or diagnostic agents.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to carboxy-terminally andcarboxy/amino-terminally phosphorylated polyamide nucleic acid (PNA)derivatives having improved properties, to their use and to agents andprocesses for preparing them.

2. Summary of the Related Art

Polyamide nucleic acids, also termed peptide nucleic acids (PNA), bindto complementary target sequences (DNA or RNA) with a higher affinitythan do natural oligonucleotides and, furthermore, have the advantage,as compared with natural DNA, that they are very stable in serum. PNAwere originally described as unnatural nucleic acid analogs in which theentire sugar-phosphate backbone is replaced with N-(2-aminoethyl)glycineunits (M. Egholm et al. (1991) Science 254, 1497-1500; WO 92/20702; M.Egholm et al. Nature (1993) 365, 566-568; P. Nielsen, (1994)Bioconjugate Chem. 5, 3-7; E. Uhlmann et al. (1998) Angewandte ChemieInt. Ed. Engl. 37, 2796-2823). The bases employed are 1) nucleobaseswhich occur naturally and are customary in nucleotide chemistry, 2)nucleobases which do not occur naturally, and 3) the prodrug forms ofthese two types of bases, that is, precursors which are only convertedinto the free base by biotransformation in the body.

PNAs have also been described in which not all the positions in thebackbone carry base residues (Greiner et al. (1999) Helv. Chim Acta 82,2151), and in which aminoethylglycine is replaced by more complex units(Uhlmann et al. (1998) Angewandte Chem. Int. Ed. 37, 2796; Falkiewicz(1999) Biochim. Pol., 46, 509-529).

The fact that the PNA backbone does not have any net charge is a featureof this class of substances that has far-reaching consequences. The factthat PNA binds to complementary DNA and RNA even at low saltconcentration (see e.g. Peptide Nucleic Acids: Protocols andApplications; Peter E. Nielsen and Michael Egholm (Edit.) HorizonScientific Press, 1999, page 3), with the Watson-Crick base pairingrules being obeyed, is ascribed to the neutral character of the PNA andthe decrease in charge repulsion which is associated therewith. For thisreason, PNA can, in principle, be used for numerous applications inwhich natural oligonucleotides or oligonucleotide derivatives wouldotherwise be employed. However, in addition to this, because of theunique binding properties, a large number of applications which are notpossible with natural oligonucleotides also ensue (see, for example:Peptide Nucleic Acids: Protocols and Applications; Peter E. Nielsen andMichael Egholm (Edit.) Horizon Scientific Press, 1999). For example, astrand invasion of double-stranded DNA has been observed when using PNA,resulting in formation of triplex structures.

Typical examples of applications for PNA include its use for inhibitinggene expression by binding, in a sequence-specific manner, to cellularDNA or RNA. “Antisense agents” are short, single-stranded nucleic acidderivatives which bind, by means of Watson-Crick base pairing, to acomplementary mRNA whose translation into the corresponding protein isto be inhibited (Uhlmann and Peyman (1990) Chem. Rev. 90, 543; Larsen etal. (1999) Biochem. Biophys. Acta 1489, 159). “Anti-gene agents” bind,by way of Hoogsteen base pairing, in the major groove of the DNA doublehelix with the formation of a triple helix, resulting in transcriptionof the genes being inhibited in a sequence-specific manner (Praseuth etal. (1999) Biochem. Biophys. Acta 1489, 181). Gene expression can alsobe specifically inhibited by so-called decoy oligomers, which mimic theregions for binding transcription factors. By treating with decoyagents, particular transcription factors can be captured in asequence-specific manner and activation of transcription therebyprevented (Mischiati et al. (1999) J. Biol. Chem. 274, 33114). Anothergroup of oligonucleotide derivatives which act intracellularly are thechimeraplasts. These are used for specific gene proof-reading(Cole-Strauss et al. (1996) Science 273, 1386-1389).

PNAs can, therefore, be used as pharmaceuticals and/or diagnostic agentsor for producing pharmaceuticals and/or diagnostic agents. For example,after having been labeled with biotin, fluorescein, or other labels, PNAcan be used as a specific hybridization probe for diagnostic purposesand in molecular biology.

Four methods have so far been described in the literature forintroducing the labeling groups (Oerum et al. (1999), in Peptide NucleicAcids: Protocols and Applications, pages 81-86; Lohse et al. (1997)Bioconjugate Chem. 8, 503). The first method is based on labeling thefree (deprotected) PNA after it has been synthesized in solution. Inthis method, the amino terminus of the PNA is reacted with an activatedcarboxylic acid or an isothiocyanate. However, additional lysineresidues are frequently introduced into the PNA, with these residuesthen being reacted with fluorescein isothiocyanate (FITC).

In the second method, the protected PNA is modified at its aminoterminus with activated carboxylic acid derivatives or isothiocyanateswhile it is still on the solid phase. This method is only suitable forlabeling groups which are stable under the conditions which pertainduring deprotection of the PNA and during its cleavage from the support.The reactive reagents which are preferably used in both cases areisothiocyanates (P. Wittung et al., (1995) FEBS Left. 375, 27) andactivated carboxylic acids, such as N-hydroxysuccinimide esters (NHS)(Oerum et al., 1999). A disadvantage of the reaction using the NHSderivatives is that it is frequently only accomplished with poor yields.For this reason, 8-amino-3,6-dioxaoctanoic acid is frequently condensed,as a linker or spacer, between the PNA and the labeling group (Oerum etal., 1999). Both linkages are effected by way of amide bonds or thioureabonds, which, as such, are, however, more likely to lead toinsolubility. Alternatively, the carboxylic acids are caused to reactusing activators which are customary in peptide chemistry, such as HBTU,TBTU or HATU.

In a third method, shown generally above, fluorescein-conjugatedmonomers are used during the synthesis of the PNA on the solid phase,with the fluorescence labeling being effected by way of an amide bond(Lohse et al. (1997) Bioconjugate Chem. 8, 503), which once again leadsto conjugates that are relatively difficult to dissolve.

A fourth method uses PNA peptide conjugates in which the peptide moietyforms a substrate for a protein kinase (Koch et al. (1995) TetrahedronLett. 36, 6933). In this way, therefore, it is not the PNA moiety whichis modified; rather, the serine residue in the peptide segment isphosphorylated enzymatically. When this method is used, therefore, it isonly possible to introduce radioactive phosphate, and not, for example,any fluorescein or biotin, into the peptide segment of the PNA-peptideconjugate. The general reaction is depicted as follows:

It is known that PNA tends to aggregate in aqueous solution, that is,under physiological conditions as well. PNA is therefore poorly solublein aqueous buffer and is then unavailable for hybridizing tocomplementary sequences. Furthermore, PNA has a high affinity forvarious materials such as SEPHADEX® (from Pharmacia), BOND ELUT® (fromVarian), or various HPLC chromatograph materials that are used inpurifying oligomers. This means that PNA can frequently only be isolatedin poor yields. It is therefore necessary to conjugate PNA with lysineor other positively charged amino acids (by way of the C terminus)(Egholm et al (1992) J. Am. Chem. Soc. 114, 1895). Guanine-rich PNAsequences have a very particular tendency to aggregate. For this reason,use of such PNA is generally discouraged (see “Guidelines for sequencedesign of PNA oligomers” in Peptide Nucleic Acids: Protocols andApplications (1999) pages 253-255). For example, relatively longfluorescein-labeled PNA oligomers are particularly difficult todissolve, with the addition of an organic solvent and heating to 50° C.being recommended.

It is particularly difficult to purify the poorly soluble lipophilic PNAderivatives. Several peaks due to PNA aggregates are frequently detectedin the HPLC. The technique of polyacrylamide (PAA) gel electrophoresis,which is frequently employed for purifying and separating nucleic acids,cannot be used for these PNA derivatives.

In the methods of derivatizing PNA which are described above, thelabeling group is always introduced by forming an amide bond or athioamide bond, with PNA derivatives being formed which are relativelydifficult to dissolve. Poorly soluble PNA derivatives are formed, inparticular, when lipophilic residues, such as fluorescein, areintroduced. Inserting labels at both ends of the PNA is technically evenmore difficult and generally leads to even poorer solubility. Inaddition, no efficient method for simultaneously derivatizing PNA at theamino and carboxy termini, in particular by means of solid phasesynthesis, has been described. Furthermore, since the labeling reactionsfrequently proceed with poor yields, there is a need in the art todevelop PNA derivatives that can be prepared in high yields, and whichshould exhibit advantageous properties, such as improved solubility,improved binding behavior, and better cellular uptake, and which, inaddition, make it possible to use efficient methods for purifying thePNA oligomers.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 a, 1 b, 2 b and 3 b show examples of terminal Z and Z′ radicals.

FIGS. 2 a and 3 a show examples of bridging X and X′ radicals.

FIGS. 4 a, 4 b, 4 c and 4 d show examples of phosphorylating reagents.

FIGS. 5 a and 5 b show examples of single (A, B) and multiple (C to E)derivatization of PNA at the N terminus.

FIG. 6 shows examples of support-bound reagents for solid phasesynthesis.

FIGS. 7, 8 and 9 show examples of synthesizing C- and N-terminallymodified PNA.

DETAILED DESCRIPTION OF THE INVENTION

According to the invention, the needs of the art are achieved byproviding PNA derivatives which carry one or more phosphoryl radicals atthe C terminus or at the C and N termini of the PNA backbone. Theinvention provides PNA derivatives that are derivitized with, amongother things, thiophosphoryl radicals, iminophosphoryl radicals, and/oroxophosphoryl radicals. The PNA derivatives of the invention can have atleast one of the phosphoryl radicals carrying one or more deprotonatablegroups, such as hydroxyl groups or mercapto groups. The phosphorylradicals can be linked to the PNA backbone by way of anoxygen-phosphorus bond, sulfur-phosphorus bond, or nitrogen-phosphorusbond, either directly or by way of a spacer. The spacer can be, but isnot necessarily, an alkanoylamide, a poly(alkoxy)carboxamide, or anamino acid. Examples of phosphoryl radicals include, but are not limitedto, phosphate, phosphonate, thiophosphate, phosphoamidate, andsubstituted phosphoryl radicals. The substituted phosphoryl radicals cancarry, where appropriate, one or more labeling groups, groups forcrosslinking, groups which promote intracellular uptake, or groups whichincrease the binding affinity of the PNA derivative for nucleic acids.

Thus, in embodiments, the invention is directed to a PNA derivativewhich carries one or more phosphoryl radicals at the C terminus or atthe C and N termini of the PNA backbone, wherein the phosphoryl radicalscomprise oxo-, thio- and imino-phosphoryl radicals, and wherein at leastone of the phosphoryl radicals carries one or more deprotonatablegroups, and wherein the phosphoryl radicals are linked to the PNAbackbone by way of an oxygen-phosphorus bond, a sulfur-phosphorus bond,or a nitrogen-phosphorus bond, either directly or by way of a spacer.

Labeling groups (labels) are understood as being groups which enable thechemical or biological activity of the PNA derivatives to be assessedqualitatively or quantitatively, for example biotin or fluorescein.Crosslinking is understood as being the formation of intramolecular orintermolecular bonds between spatially adjacent functionalities. Anexample of a group for crosslinking is the psoralen group.

In general, the invention relates to PNA derivatives of Formula I

wherein

-   q is 0 or 1,-   D′ is hydroxyl, mercapto, amino, alkylamino, or acylamino, such as    acetylamino,-   V is oxygen, sulfur, or NR₁,-   V′ is, independently of any other V′, oxygen, sulfur, NR₁.    U—(CR₃R₄)_(u′)—C(O)—NH, or U—(CH₂CH₂O)_(u′)—CH₂—C(O)—NH,-   U is, independently of any other U, oxygen, sulfur, or NH,-   u′ is, independently of any other u′, from 1 to 10, such as from 1    to 4, for example, 1,-   W and W′ are, independently of each other, oxygen, sulfur, or NR₁,-   Y and Y′ are, independently of each other, hydroxyl, mercapto,    oxyanion, thioate, or NR₁R₂,-   X and X′ are, independently of each other, U—(C₂-C₂₂-alkanediyl)-U,    U—(CH₂CH₂—O)_(u′), a labeling group, a group for crosslinking, a    group which promotes intracellular uptake, or a group which    increases the binding affinity of the PNA derivative for nucleic    acids, for example a bifunctional fluorescein, rhodamine, TAMRA,    biotin, pyrene, dinitrophenyl, cholesteryl, acridine, adamantyl,    vitamin E, cyanine dye, dabcyl, edans, lexitropsin, psoralen,    BODIPY, ROX, R6G or digoxygenin radical,-   Z and Z′ are, independently of each other,    -   hydroxyl,    -   mercapto,    -   oxyanion,    -   thioate,    -   NR₁R₂,    -   C₁-C₂₂-alkyl,    -   C₁-C₈-arylalkyl,    -   C₁-C₂₂-alkyl-U,    -   C₁-C₈-arylalkyl-U,    -   hydroxy-C₁-C₁₈—U,    -   aminoalkyl-U,    -   mercaptoalkyl-U,        -   a group of the formula R₇(CH₂CH₂—O)_(m′), wherein R₇ is            hydroxyl, amino, or C₁-C₂₂-alkoxy, and m′ is from 1 to 100,            such as from 2 to 10,    -   a labeling group,    -   a crosslinking group,    -   a group which promotes intracellular uptake,    -   or a group which increases the binding affinity of the PNA        derivative for nucleic acids, for example a monofunctional or        bifunctional fluorescein, rhodamine, TAMRA, biotin or a biotin        derivative, pyrene, dinitrophenyl, cholesteryl, acridine,        adamantyl, vitamin E, cyanine dye, dabcyl, edans, lexitropsin,        psoralen, BODIPY, ROX, R6G or digoxygenin radical,-   R₁ and R₂ are, independently of each other, a radical consisting of    hydrogen or C₁-C₆-alkyl, for example hydrogen,-   R₃ and R₄ are, independently of each other, a radical consisting of    hydrogen, or C₁-C₆-alkyl, or the radical of an amino acid side    chain, for example hydrogen, it being possible for adjacent radicals    R₃ and R₄ in V′ to also form a C₅-C₈-cycloalkyl ring,-   n is from 0 to 10, such as from 0 to 3,-   m is from 0 to 10, such as from 0 to 3,-   with the proviso that at least one of the Y, Y′, Z, or Z′ radical is    hydroxyl, mercapto, oxyanion, or thioate;-   and wherein {POLY} is described by Formula II,

wherein {BLOCK} is, independently of any other [BLOCK},Formula IIIA,

Formula IIIB (Greiner et al. (1999) Helv. Chim Acta 82, 2151),

or Formulae IV A to IV G (Uhlmann et al. (1998) Angewandte Chem. Int.Ed. 37, 2796; Falkiewicz (1999) Biochim. Pol., 46, 509-529),

wherein each {BLOCK} building block can be different;

-   and wherein-   Z″ is from 0 to 100, such as 1-20, for example 4-15,-   G is selected from the groups (CR₅R₆)_(u′), C(O)NH—(CR₁R₂)_(t′), or    C(O)NH—(CH₂CH₂O)_(u′)—CH₂CH₂, wherein u′ has the above-mentioned    meaning and t′ is from 2 to 10, for example 6,-   A is, independently of any other A, a group (CR₁R₂)_(S), wherein s    is from 1 to 3, for example 1,-   B is, independently of any other B, either    -   an aromatic radical which optionally possesses heteroaromatic        character, hydrogen, hydroxyl, or C₁-C₁₈-alkyl, or    -   a nucleobase which occurs naturally, and is customary in        nucleotide chemistry, or which does not occur naturally, or its        prodrug form,    -   with the proviso that at least one B radical is a nucleobase,-   D is, independently of any other D, a group (CR₃R₄)_(t), wherein t    is from 2 to 10, such as from 2 to 4, for example 2,-   E is, independently of any other E, a group (CR₅R₆)_(u′), wherein    adjacent R₅ and R₆ radicals can also form a C₅- to C₈-cycloalkyl    ring or a spiro compound,-   R₅ and R₆ are, independently of each other, a radical consisting of    hydrogen, C₁-C₆-alkyl, or the radical of an amino acid side chain,    for example hydrogen,-   wherein u′, R₁, R₂, R₃, and R₄ are as defined above.

In addition, the invention relates to physiologically tolerated salts ofthe PNA derivatives of Formula I. Physiologically tolerated salts aredescribed, for example, in Remington's Pharmaceutical Science (1985)Mack Publishing Company, Easton, Pa., USA, page 1418. In embodiments,the salts are ammonium salts, trialkylammonium salts, alkali metal salts(such as sodium salts and potassium salts), and alkaline earth metalsalts (such as magnesium salts and calcium salts). In embodiments, thesalts are sodium salts.

A surprising, positive effect which was found was that the introductionof a phosphoryl radical, for example as phosphate or else in the form ofa lipophilic derivatization (e.g. as a hexadecyl phosphodiester)increases the affinity of the PNA for complementary DNA or RNA. Thiseffect was unexpected since the strong bonding of PNA to complementaryDNA or RNA was attributed to the neutral character of the PNA and thereduced charge repulsion which was associated therewith (e.g. PeptideNucleic Acids: Protocols and Applications; Peter E. Nielsen and MichaelEgholm (Edit.) Horizon Scientific Press, 1999, page 3).

The biotin was introduced particularly efficiently by way of aphosphoryl radical. When used as hybridization probes, the biotinylatedPNA of Formula I (X, X′, Z, and/or Z′=biotin radical) displayed betterbinding properties and fewer spurious, nonspecific background effectsthan did corresponding biotinylated DNA probes.

In contrast to the uncharged PNA, the PNA derivatives of Formula Iaccording to the invention can also migrate in an electric field,thereby making it possible to microlocate them and concentrate them onimmobilized complementary nucleic acid derivatives. In the case ofpolyanionic oligonucleotides, the use of an electrical field formicrolocation and concentration has already been described for rapidlydetermining base mismatches (Sosnowski et al. (1997) Proc. Natl. Acad.Sci. U.S.A. 94, 1119).

The hydroxy or mercapto substituents of the phosphoryl radicals of theDNA derivatives according to the invention can be deprotonated in a pHrange of from 4.5 to 14. In embodiments, they are deprotonated in a pHrange of from 6.5 to 12, such as from 6.5 to 9. The property of theionizability of the phosphoryl radicals can advantageously be exploitedfor purifying the compounds of Formula I. On the one hand, the compoundsof Formula I can be purified by electrophoresis, for examplepolyacrylamide gel electrophoresis (PAGE). On the other hand, it is alsopossible to purify them using anion exchangers. In the latter case, thedesired products can be eluted by using a salt gradient, for example asodium chloride gradient. They can also be eluted by using a pHgradient. The PNA derivatives of Formula I according to the inventioncan be simply and efficiently purified using anion exchangers. It wasfound that the uncharged byproducts are not retarded on the anionexchanger, whereas the charged product adhered to the column. Afterwashing with water, it was possible to isolate the desired product inpure form using acetic acid or a sodium chloride solution. The anionexchangers employed can be strong anion exchangers or mixed-mode phases,such as OASIS MAX® (Waters GmbH, Eschborn).

It was furthermore found that the compounds of Formula I according tothe invention are, in general, more readily soluble in aqueous mediumthan are the corresponding PNA oligomers which do not possess thephosphoryl radical. This is particularly apparent in the form of agreatly improved solubility in aqueous medium in the case of thelipophilic derivatives, such as the fluorescein derivatives or thehexadecyl derivatives.

The invention relates, in embodiments, to PNA derivatives in which A andE of Formula IIIA and/or Formula IIIB are CH₂. The invention furthermorerelates, in embodiments, to PNA derivatives in which D substituents ofFormula IIIA and/or Formula IIIB are (CH₂)₂. In embodiments, theinvention relates to PNA derivatives of Formula I in which W and W′ areoxygen. In embodiments, the invention relates to PNA derivatives ofFormula I in which Y and Y′ are hydroxyl or oxyanion. In embodiments,the invention relates to PNA derivatives of Formula I in which V and V′are oxygen radicals.

Non-exclusive examples of natural bases are adenine, cytosine,5-methylcytosine, guanine, thymine, and uracil. Non-exclusive examplesof unnatural bases are purine, 2,6-diaminopurine, N⁴N⁴-ethanocytosine,N⁶N⁶-ethano-2,6-diaminopurine, 5-(C₃-C₆)-alkynyluracil,5-(C₃-C₆)-alkynylcytosine, 5-(1-propargylamino)uracil,5-(1-propargylamino)cytosine, phenoxazine, 9-aminoethoxyphenoxazine,5-fluorouracil or pseudoisocytosine, 5-(hydroxymethyl)uracil,5-aminouracil, pseudouracil, dihydrouracil, 5-(C₁-C₆)-alkyluracil,5-(C₁-C₆)-alkylcytosine, 5-(C₂-C₆)-alkenylcytosine, 5-fluorocytosine,5-chlorouracil, 5-chlorocytosine, 5-bromouracil, 5-bromocytosine,7-deazaadenine, 7-deazaguanine, 8-azapurine, and 7-deaza-7-substitutedpurines.

In the case of PNA derivatives which only carry a phosphoryl radical atthe C terminus (and for which q is 0), the N terminus can be linked to apeptide sequence. Suitable peptide sequences are those which optimizethe organ distribution or the cellular location of the PNA, such astransportan, insulin-like growth factor, nuclear localization signals,or other carrier sequences (Larsen et al. (1999) Biochim. Biophys. Acta159-166). The peptide can also be used as an affinity tag, like, forexample, a (His)₆ chain.

The present invention enables the X, X′, Z, and Z′ radicals to be variedbroadly (non-limiting examples are given in FIGS. 1 a, 1 b, 2 a, 2 b, 3a, and 3 b) and thereby makes it possible to introduce differentspecific functional features into the PNA.

One embodiment of Z or Z′ is a C₁- to C₂₂-alkyl radical. In otherembodiments, Z or Z′ is a C₁- to C₂₂-alkoxy radical, for example aC₁₆-alkoxy radical. Other suitable radicals include, but are not limitedto, hydroxy-(C₁-C₁₈-alkoxy) radicals, such as HO(CH₂)₃₋₁₂O. Inembodiments, Z or Z′ is an aminoalkoxy radical, such as a 6-aminohexoxyor 5-aminopentoxy radical. In embodiments, Z or Z′ is a radical of theformula R₇(CH₂CH₂—O)_(m), wherein R₇ is hydroxyl, amino, orC₁-C₂₂-alkoxy. In embodiments, R₇ is hydroxyl. In embodiments, m is from0 to 100. In embodiments, m is from 2 to 10. In embodiments, Z or Z′ isHO(CH₂CH₂—O)₂, HO(CH₂CH₂—O)₆, or H₂N—(CH₂CH₂—O)₂. In other embodiments,Z or Z′ is a mercaptoalkoxy radical, such as 6-mercaptohexyloxy.

In another embodiment, Z or Z′ comprises a fluorescent group, such asfluorescein, rhodamine, TAMRA, or a cyanine dye. Non-limiting examplesof suitable fluorescent groups can be found in FIGS. 1 a to 3 b. Inembodiments, Z is biotin or a biotin derivative. In yet otherembodiments, Z is dabcyl, psoralen, acridine, DNP, cholesterol (see, forexample, FIGS. 1 b and 2 b), BODIPY, ROX or R6G radicals (Su-Chun Hunget al. (1998) Analytical Biochemistry 255, 32-38), or digoxygenin(Tarrason et al., Methods in Enzymology (1999) Vol. 313, 257-268).

In addition to this, Z or Z′ can be a group consisting of amonofunctional or a bifunctional fluorescein, rhodamine, TAMRA, biotin,pyrene, dinitrophenyl, cholesteryl, acridine, adamantyl, vitamin E,cyanine dye, dabcyl, edans, lexitropsin, or psoralen radical.Monofunctional end groups are listed by way of example in FIGS. 1 a, 1b, 2 a and 3 a, while bifunctional bridging groups are listed by way ofexample in FIGS. 2 b and 3 b. In another embodiment, n and/or m,independently of each other, are 0, i.e. the PNA moiety carries in eachcase only one phosphoryl radical on the N and/or on the C terminus.

In an embodiment, X or X′ is U—(C₂-C₂₂-alkanediyl)-U, such asO—(C₂-C₂₂-alkanediyl)-O. For example X or X′ can be O—(CH₂)₂₋₆—O. Inanother embodiment, X or X′ is a group of the Formula U—(CH₂CH₂—O)_(u′),wherein u′ is from 1 to 10, such as from 1 to 6. In embodiments, U canbe oxygen. In a further embodiment, X or X′ comprises a fluorescentgroup such as fluorescein, rhodamine, TAMRA, or a cyanine dye, forexample Cy3® (from Amersham Pharmacia Biotech). Exemplary bifunctionalgroups can be found in FIGS. 2 a and 3 a. In embodiments, X or X′ isbiotin. Other groups which are suitable are dabcyl, psoralen, acridine,DNP, cholesterol, BODIPY, lexitropsin, digoxygenin, and ROX and R6Gradicals.

The different radicals for X, X′, Z, and Z′ in Formula I can fulfilldifferent functions. The fluorescein radicals have far-reachingapplications in DNA sequencing and signal amplification or as markersfor determining the cellular uptake of PNA. The cyanine dye radicals(Cy3® and Cy5®) give a substantially more intense and longer-lastingfluorescence signal than does fluorescein itself. The psoralen radicalcan be employed for crosslinking with complementary nucleic acids. Theacridine radical is an effective intercalator and can thereby augmentthe binding affinity of the PNA. Biotin, acridine, and psoralenderivatives can also be used for antisense experiments. In addition,hexadecyloxy and cholesterol derivatives can be used for increasing theability of the PNA to traverse membranes. DNP-labeled compounds ofFormula I can be detected using anti-DNP antibodies. Aminoalkoxyradicals can be used for coupling on other groups, for examplelexitropsin (cf. Example 17; PNA-16). In a similar way, mercaptoalkoxygroups can also be used for further derivatization.

The invention furthermore relates to the use of the PNA derivatives ofFormula I as pharmaceuticals. These pharmaceuticals can be used forpreventing and/or treating diseases which are accompanied by theexpression or overexpression of particular genes. The inventionfurthermore relates to the use of PNA derivatives as diagnostic agents.These diagnostic agents can be used for diagnosing diseases at an earlystage.

When being employed as pharmaceuticals or diagnostic agents, the PNAderivatives of Formula I can be used as antisense agents, anti-geneagents, decoy agents, and chimeraplast agents, depending on theirsequence.

The PNA derivatives according to the invention are used, for example,for producing pharmaceuticals for treating diseases in which definedgenes are the cause, or are involved, as a result of theiroverexpression. These pharmaceuticals can, for example, be used fortreating diseases which are provoked by viruses, for example by CMV,HIV, HSV-1, HSV-2, influenza, VSV, hepatitis B, or papilloma viruses,with the corresponding virus sequence being the target.

Antisense PNA derivatives according to the invention which are activeagainst these targets have, for example, the following base sequences:

-   a) against CMV, for example

SEQ ID NO:1 5′-G C G T T T G C T C T T C T T C T T G C G-3′

-   b) against HIV, for example

SEQ ID NO:2 5′-A C A C C C A A T T C T G A A A A T G G-3′ SEQ ID NO:35′-A G G T C C C T G T T C G G G C G C C A-3′

-   c) against HSV-1, for example

SEQ ID NO:4 5′-G C G G G G C T C C A T G G G G G T C G-3′.

Such pharmaceuticals are also suitable, for example, for treatingcancer. In this connection, in embodiments, sequences are used which aredirected against targets which are responsible for the carcinogenesis orthe growth of a cancer, such as by inhibiting telomerase (E. Matthes etal. (1999) Nucleic Acids Res. 27, 1152). Additional targets of thisnature include, but are not limited to:

-   1) Nuclear oncoproteins, such as for example, c-myc, N-myc, c-myb,    c-fos, c-fos/jun, PCNA, and p120;-   2) Cytoplasmic/membrane-associated oncoproteins, such as for    example, EJ-ras, c-Ha-ras, N-ras, rrg, bcl-2, cdc-2, c-raf-1, c-mos,    c-src, c-abl, and c-ets;-   3) Cell receptors, such as for example, EGF receptor, Her-2, c-erbA,    VEGF receptor (KDR-1), retinoid receptors, protein kinase regulatory    subunit, c-fms, Tie-2, c-raf-1 kinase, PKC-alpha, and protein kinase    A (R1 alpha); and-   4) Cytokines, growth factors, and extracellular matrix, such as for    example, CSF-1, IL-6, IL-1a, IL-1b, IL-2, IL-4, IL-6, IL-8, bFGF,    VEGF, myeloblastin, and fibronectin.

Antisense PNA derivatives which are active against such targets have,for example, the following base sequences:

-   a) against c-Ha-ras, for example

5′-C A G C T G C A A C C C A G C-3′ SEQ ID NO:5 5′-T A T T C C G T C AT-3′ SEQ ID NO:6 SEQ ID NO:7 5′-T T C C G T C A T C G C T C C T C A G GG G-3′

-   b) bFGF, for example

5′-G G C T G C C A T G G T C C C-3′ SEQ ID NO:8

-   c) c-myc, for example

SEQ ID NO:9 5′-G G C T G C T G G A G C G G G G C A C A C-3′ 5′-A A C G TT G A G G G G C A T-3′ SEQ ID NO:10

-   d) c-myb, for example

SEQ ID NO:11 5′-G T G C C G G G G T C T T C G G G C-3′

-   e) c-fos, for example

SEQ ID NO:12 5′-C G A G A A C A T C A T C G T G G-3′ SEQ ID NO:13 5′-G GA G A A C A T C A T G G T C G A A A G-3′ SEQ ID NO:14 5′-C C C G A G A AC A T C A T G G T C G A A G-3′ SEQ ID NO:15 5′-G G G G A A A G C C C G GC A A G G G G-3′

-   f) p120, for example

SEQ ID NO:16 5′-C A C C C G C C T T G G C C T C C C A C-3′

-   g) EGF receptor, for example

SEQ ID NO:17 5′-G G G A C T C C G G C G C A G C G C-3′ SEQ ID NO:18 5′-GG C A A A C T T T C T T T T C C T C C-3′

-   h) p53 tumor suppressor, for example

SEQ ID NO:19 5′-G G G A A G G A G G A G G A T G A G G-3′ SEQ ID NO:205′-G G C A G T C A T C C A G C T T C G G A G-3′

-   i) bcl-2, for example

SEQ ID NO:21 5′-T C T C C C A G C G T G C G C C A T-3′

-   j) VEGF, for example

SEQ ID NO:22 5′-G C G C T G A T A G A C A T C C A T G-3′ 5′-G G A G G CC C G A C C-3′ SEQ ID NO:23 5′-G G T T T C G G A G G C-3′ SEQ ID NO:245′-T G G T G G A G G T A G-3′ SEQ ID NO:25 5′-G C A T G G T G G A G G-3′SEQ ID NO:26 5′-T T G G C A T G G T G G-3′ SEQ ID NO:27 5′-G C C T G G GA C C A C-3′ SEQ ID NO:28 5′-C A G C C T G G G A C C-3′ SEQ ID NO:295′-T G C A G C C T G G G A-3′ SEQ ID NO:30 5′-G T G C A G C C T G G G-3′SEQ ID NO:31 5′-G G T G C A G C C T G G-3′ SEQ ID NO:32 5′-A T G G G T GC A G C C-3′ SEQ ID NO:33 5′-G G C T T G A A G A T G-3′ SEQ ID NO:345′-G C A G C C C C C G C A-3′ SEQ ID NO:35 5′-G C A G C A G C C C C C-3′SEQ ID NO:36

-   k) c-raf kinase, for example

SEQ ID NO:37 5′-T C C C G C C T G T G A C A T G C A T T-3′

-   l) PKC-alpha, for example

SEQ ID NO:38 5′-G T T C T C G C T G G T G A G T T T C A-3′

-   m) protein kinase A, for example

SEQ ID NO:39 5′-G C G T G C C T C C T C A C T G G C-3′.

Pharmaceuticals comprising PNA derivatives of Formula I are furthermoresuitable, for example, for treating diseases which are effected byintegrins or cell-cell adhesion receptors, for example by VLA-4, VLA-2,ICAM, VCAM, or ELAM.

Antisense PNA derivatives which are active against such targets have,for example, the following base sequences:

-   a) VLA-4, for example

SEQ ID NO:40 5′-G C A G T A A G C A T C C A T A T C-3′

-   b) ICAM-1, for example

SEQ ID NO:41 5′-G C C C A A G C T G G C A T C C G T C A-3′ SEQ ID NO:425′-C C C C C A C C A C T T C C C C T C T C-3′ SEQ ID NO:43 5′-C T C C CC C A C C A C T T C C C C T C-3′ SEQ ID NO:44 5′-G C T G G G A G C C A TA G C G A G G-3′

-   c) ELAM-1, for example

SEQ ID NO:45 5′-A C T G C T G C C T C T T G T C T C A G G-3′ SEQ IDNO:46 5′-C A A T C A A T G A C T T C A A G A G T T C-3′

-   d) Integrin alpha(V), for example for example

SEQ ID NO:47 5′-G C G G C G G A A A A G C C A T C G-3′.

Pharmaceuticals comprising PNA derivatives of Formula I are alsosuitable, for example, for preventing restenosis. In this connection, itis possible to use PNA sequences which are directed against targetswhich are responsible for proliferation or migration. Examples of suchtargets are:

-   1) Nuclear transactivator proteins and cyclins, such as for example    c-myc, c-myb, c-fos, c-fos/jun, cyclins, and cdc2 kinase;-   2) Mitogens or growth factors, such as for example PDGF, bFGF, VEGF,    EGF, HB-EGF, and TGF-β; and-   3) Cell receptors, such as for example bFGF receptor, EGF receptor,    and PDGF receptor.

Antisense PNA derivatives which are active against such targets have,for example, the following base sequences:

-   a) c-myb, for example

SEQ ID NO:48 5′-G T G T C G G G G T C T C C G G G C-3′

-   b) c-myc, for example

5′-C A C G T T G A G G G G C A T-3′ SEQ ID NO:49

-   c) cdc2 kinase, for example

SEQ ID NO:50 5′-G T C T T C C A T A G T T A C T C A-3′

-   d) PCNA (proliferating cell nuclear antigen of rat), for example

SEQ ID NO:51 5′-G A T C A G G C G T G C C T C A A A-3′.

PNA derivatives can likewise be used for treating vitiligo and otherdepigmentation diseases or depigmentation disturbances (e.g. of theskin, the hair, and the eyes), such as albinism and psoriasis, or fortreating asthma, with expression of the adenosine A1 receptor, theadenosine A3 receptor or the bradykinin receptor, or of IL-13, beinginhibited using suitable antisense agents. An example of such a basesequence is:

SEQ ID NO:52 5′-G A T G G A G G G C G G C A T G G C G G G-3′.

Pharmaceuticals that comprise a PNA derivative of Formula I can be used,for example, in the form of pharmaceutical preparations which can beadministered orally, for example in the form of tablets, coated tablets,hard or soft gelatin capsules, solutions, emulsions, or suspensions.They can also be administered rectally, e.g. in the form ofsuppositories, or parenterally, e.g. in the form of solutions forinjection. In order to produce pharmaceutical preparations, thesecompounds can be processed in therapeutically inert organic andinorganic excipients. Examples of such excipients for tablets, coatedtablets and hard gelatin capsules are lactose, cornstarch or derivativesthereof, tallow and stearic acid or salts thereof. Suitable excipientsfor preparing solutions include, but are not limited to, water, polyols,sucrose, invert sugar, and glucose. Suitable excipients for injectionsolutions include, but are not limited to, water, alcohols, polyols,glycerol, and vegetable oils. Suitable excipients for suppositoriesinclude, but are not limited to, vegetable oils and hydrogenated oils,waxes, fats, and semiliquid polyols. The pharmaceutical preparations canalso comprise preservatives, solvents, stabilizers, wetting agents,emulsifiers, sweeteners, dyes, flavorants, salts for altering theosmotic pressure, buffers, coating agents, antioxidants and, whereappropriate, other therapeutically active compounds. The identity andamount of excipient, carrier, and/or additive should conform to thepractices known to those of skill in the pharmaceutical art. Techniquesfor preparation of pharmaceuticals according to the present inventionare well known to those of skill in the art and are well within theskill of those artisans. Accordingly, the techniques need not bedetailed here. Treatment regimens (e.g., number of doses per unit time,length of treatment, etc.) should conform to the practices known tothose of skill in the pharmaceutical art.

Administration forms include, but are not limited to, topicalapplications; local applications, for example using a catheter or byinhalation; injections or infusions; and oral administration. Forinjection, the PNA derivatives of Formula I are formulated in a liquidsolution, such as in a physiologically acceptable buffer (for exampleHank's solution or Ringer's solution). However, the oligonucleotides canalso be formulated in solid form and dissolved or suspended before use.Suitable doses for systemic administration are from about 0.01 mg/kg toabout 50 mg/kg of bodyweight and per day

The invention furthermore relates to pharmaceutical preparations whichcomprise PNA derivatives of Formula I and/or their physiologicallytolerated salts in addition to pharmaceutically acceptable excipientsand/or additives.

The PNA derivatives of Formula I and/or their physiologically toleratedsalts can be administered to animals, including mammals. In embodiments,the mammal is a human. In embodiments, the mammal is a feline, such as acat, or a canine, such as a dog. In embodiments, the mammal is anequine, such as a horse; an ovine, such as a cow or steer; a porcine,such as a pig; or an ovine, such as a sheep.

In embodiments, the PNA derivatives of Formula I and/or theirphysiologically acceptable salts are prepared as pharmaceuticals. Inembodiments, they are prepared on their own as pharmaceuticals or theyare prepared in mixtures with each other as pharmaceuticals. In otherembodiments, they are prepared in the form of pharmaceuticalpreparations which permit topical, percutaneous, parenteral, or enteraluse and which comprise, as the active constituent, an effective dose ofat least one PNA derivative together with at least one customary,pharmaceutically acceptable excipient and/or additive. The preparationscan comprise from about 0.1 to 90% by weight of the therapeuticallyactive compound. A topical application, for example in the form ofointments, lotions, tinctures, emulsions, or suspensions, is suitablefor treating skin diseases.

As discussed above, the pharmaceutical preparations are produced in amanner known to those of skill in the art (see, for example, Remington'sPharmaceutical Sciences, Mack Publ. Co., Easton, Pa.), withpharmaceutically inert inorganic and/or organic excipients being used.It is possible, for example, to use lactose, cornstarch and/orderivatives thereof, tallow, stearic acid and/or its salts, etc., forproducing pills, tablets, coated tablets, and hard gelatin capsules,among other things. Non-exclusive examples of excipients for softgelatin capsules and/or suppositories are fats, waxes, semisolid andliquid polyols, natural and/or hydrogenated oils, etc. Suitableexcipients for producing solutions and/or syrups are, for example,water, sucrose, invert sugar, glucose, polyols, etc. Suitable excipientsfor producing solutions for injection include, but are not limited to,water, alcohols, glycerol, polyols, vegetable oils, etc. Suitableexcipients for microcapsules, implants and/or rods include, but are notlimited to, copolymers consisting of glycolic acid and lactic acid.Liposome formulations which are known to the skilled person (N. Weiner,Drug Develop Ind Pharm 15 (1989) 1523; “Liposome Dermatics, SpringerVerlag 1992), for example HVJ liposomes (Hayashi, Gene Therapy 3 (1996)878) are also suitable. Dermal application can also be effected, forexample, using ionophoretic methods and/or using electroporation.

In addition to the active compounds and excipients, a pharmaceuticalpreparation can also contain additives, such as fillers, extenders,disintegrants, binders, glidants, wetting agents, stabilizers,emulsifiers, preservatives, sweeteners, dyes, flavorants or aromatizingagents, thickeners, diluents, and buffering substances, and,furthermore, solvents and/or solubilizing agents and/or agents forachieving a sustained release effect, and also salts for altering theosmotic pressure, coating agents and/or antioxidants. They can alsocomprise two or more different PNA derivatives of Formula I and/or theirphysiologically tolerated salts and also, furthermore, in addition to atleast one PNA derivative of Formula I, one or more differenttherapeutically active substances. The dose can vary within wide limitsand is to be adjusted to the individual circumstances in each individualcase. As mentioned above, regulating dosage is well within the abilitiesof those of skill in the art.

The invention furthermore relates to the use of PNA derivatives ofFormula I as diagnostic agents, in particular as aids in DNA diagnosisand in molecular biology (see, for example: Peptide Nucleic Acids:Protocols and Applications; Peter E. Nielsen and Michael Egholm (Edit.)Horizon Scientific Press, 1999). In DNA diagnosis, gene probes, alsotermed DNA probes or hybridization probes, play an important role in thesequence-specific detection of particular genes. In general, a geneprobe consists of a recognition sequence and one or more suitablelabeling groups (labels). The specificity with which a target sequencein an analytical sample is identified by means of hybridization with acomplementary gene probe is determined by the recognition sequence andits chemical structure. This technique can also be applied to PNA. Ascompared with oligonucleotides having a natural structure, PNA has theadvantage that it has a higher affinity for the target sequence and agreater ability to discriminate between bases.

In an embodiment, the PNA are used in a method for detecting a nucleicacid of interest. In the method, the PNA is labeled with a detectablelabel, wherein the PNA derivative comprises a base sequence thathybridizes with at least one sequence present in the nucleic acid ofinterest under selected conditions (for example, stringency conditionsthat permit specific hybridization). The labeled PNA is combined with asample suspected of containing the nucleic acid of interest underconditions where specific binding of the PNA derivative to the nucleicacids in the sample can occur. Specific binding of the PNA derivativeand nucleic acids present in the sample can then be detected usingtechniques suitable for the label and known to those of skill in theart. Specific binding indicates the presence of the nucleic acid ofinterest in the sample.

In embodiments, the nucleic acid is a viral nucleic acid. Inembodiments, the nucleic acid is a nucleic acid from a microorganism(e.g., a bacterium).

The use of the compounds of Formula I therefore also relates to in-situhybridization and fluorescence in-situ hybridization (FISH). In-situhybridization can also be used, for example, for detectingmicroorganisms and viruses (Just et al. (1998) J. Vir. Method. 73,163-174).

Another application of the compounds of the invention relates todetecting and quantifying nucleic acids. Methods for performing suchassays can follow along the steps provided above, with the additionalstep of quantifying the detected nucleic acid using techniques known tothose of skill in the art, for example, comparison to concentrationstandard curves, extrapolation based on extinction coefficients, etc. Inaddition, for quantitation, use can be made of array technology(Strother et al. J. Am. Chem. Soc. (2000) 122, 1205-1209; Niemeyer etal., Angew. Chem. (1999) 111, 3039-3043; Pirrung (1997) Chem. Rev. 97,473-488), which provides high sample throughput and a high degree ofsensitivity. In embodiments, the PNA probes are fixed on a suitablesupport or PNA chip. To achieve this, PNA can be synthesized asdescribed in the examples and subsequently fixed onto the support or PNAchip. Alternatively, the PNA can be prepared directly on the support.Another application is the use of the compounds of Formula I asbiosensors for detecting nucleic acids (Wang et al (1996) J. Am. Chem.Soc. 118, 7667). The use of PNA of the Formula I possessing an affinitylabel, such as histidyl-PNA, is another application for purifyingnucleic acids (Oerum et al. (1999), in Peptide Nucleic Acids: Protocolsand Applications).

The two phosphoryl radicals at the amino terminus and the carboxyterminus can fulfill different functions. For example, the aminoterminus can be substituted lipophilically in order to increase the celluptake, with a fluorescein residue being located at the carboxy terminusfor the purpose of detecting the improved cell uptake (cf. PNA-6 inExample 7). Other examples will be apparent to those of skill in the artbased on the substituents suitable for inclusion in the PNA derivativesof the invention, as disclosed herein.

The doubly derivatized compounds of Formula I are also suitable for useas so-called “molecular beacons” (Li et al. (2000) Angew. Chemie 112,1091-1094), which only emit a fluorescence signal in association withbinding to a complementary nucleic acid. In these beacons, one end ofthe PNA, for example the amino terminus, is provided with a fluorescentlabel whereas the other end, for example the carboxy terminus, isprovided with a quencher. The opposite case, in which the N terminuscarries a quencher and the C terminus carries a fluorescent label, isalso possible. This results in the fluorescence signal being suppressedas long as the doubly labeled PNA derivative does not bind to acomplementary nucleic acid. It is only in association with binding thatthe fluorescent residue (e.g. edans) and the quencher (e.g. dabcyl)become spatially separated from each other, resulting in a fluorescencesignal being emitted (Sokol et al. (1998) Proc. Natl. Acad. Sci. 95,11538).

The PNA backbone can be synthesized using the methods described in theliterature, for example using the tert-butyloxycarbonyl(BOC),9-fluorenylmethoxycarbonyl (Fmoc), or monomethoxytrityl (Mmt) protectinggroup strategy (Peptide Nucleic Acids: Protocols and Applications; PeterE. Nielsen and Michael Egholm (Edit.) Horizon Scientific Press, 1999).In embodiments, the Mmt protecting group is used for temporarilyprotecting the amino function of the aminoethylglycine and base-labileprotecting groups on the heterocyclic nucleobases (D. Will et al. (1995)Tetrahedron 51, 12069; Breipohl et al. (1997) Tetrahedron 53,14671-14686). Examples of monomeric building blocks are compounds of theFormulae V to V D (below), with A, B, D, E, u′ and V′ having themeanings defined above. PG can be an amino-protecting group such asbenzoyl, anisoyl-, isobutyroyl-, acetyl-, or tert-butylbenzoyl (Breipohlet al. (1997) Tetrahedron 53, 14671-14686). TR can be an acid-labileprotecting group such as dimethoxytrityl (Dmt) (for V′═O and S) or Mmt(for V′═NH).

After the PNA backbone has been constructed, the free amino function ofthe N terminus can be reacted directly with an appropriatephosphorylating reagent, for example to give the correspondingphosphoramidate (V′═NR₁ in Formula I).

The phosphoryl radicals can be introduced using the reagents which arecustomarily employed in nucleotide chemistry. There are a large numberof phosphorylating reagents available which can be used for preparingthe compounds of the Formula I. A non-limiting selection of the reagentsis shown in FIGS. 4 a to 4 d, with the invention not, however, beingrestricted to these special derivatives. Appropriately modifiedsupports, in particular CPG supports for solid phase synthesis, are usedfor the carboxy-terminal modification. Non-limiting examples of suchsupports are listed in FIG. 6.

The phosphorylating reagents employed can be the reagents which arecustomary in nucleotide chemistry (Glen Research Corporation, Sterling,Va. 20164, U.S.A.; FIGS. 4 a to 4 d) and which react, for example, inaccordance with the phosphoramidite method, the H-phosphonate method orthe phosphotriester method (E. Sonveaux (1986) Bioorganic Chemistry 14,274; S. L. Beaucage and R. P. Iyer (1993) Tetrahedron 49, 1925; E.Uhlmann and A. Peyman (1990) Chem. Rev. 90, 543). The wide variety ofpossible modifications is determined by the large number of knownphosphorylating reagents and appropriately derivatized supports, inparticular of controlled pore glass (CPG) supports. TENTAGEL® (from RappPolymers GmbH, Tübingen) and aminomethylpolystyrene can be used as solidsupports.

In principle, all the reagents which are known in nucleotide chemistryare suitable for introducing the phosphoryl function. Non-exclusive,exemplary reagents are the following reagents of the Formulae VI A, VIB, VI C and VI D

wherein K is halogen (for example, Cl), triazolyl, imidazolyl, ordialkylamino. W can have the above-mentioned meaning or the meaning ofW′, and Z can have the above-mentioned meaning or the meaning of X, X′,or Z′, with reactive groups being appropriately protected.

For example, the hydroxyl groups of the fluorescein-phosphoramidite 3(FIG. 4 a) can be protected by esterifying with pivalic acid.

The compounds of Formula VI are only to be regarded as being examples ofsuch reagents, which react, where appropriate, in the added presence ofother auxiliary reagents such as bases, acids, or condensing reagents.In embodiments, the reagents of Formula VI A are those which react inaccordance with the phosphoramidite method (Beaucage and Iyer, 1993).These reagents are reacted as the phosphorus (III) compound andsubsequently oxidized. If, for example, the oxidation is carried outusing iodine/water/pyridine or tert-butyl hydroperoxide, the phosphorylderivatives (W═O) are then obtained. If, on the other hand, theoxidation is carried out using elemental sulfur or Beaucage reagent, thecorresponding thiophosphoryl compound (W═S) is then obtained.

Among the reagents (FIGS. 4 a to 4 d), are also to be found“bifunctional reagents” which, because of the possession of a secondfunction, which is initially protected, can be caused to react severaltimes. The phosphoramidites 4, 6, and 8 to 13 are examples of suchbifunctional reagents. In this connection, it can be a matter of themultiple conjugation of a reagent or else of successive reaction withdifferent reagents. Thus, for example, the fluorescein-phosphoramidite 3can only be caused to react once. By comparison, thefluorescein-phosphoramidite 4 possesses a Dmt group-protected hydroxylfunction which can be reacted once again with a phosphorylating reagentafter the Dmt group has been eliminated. In this way, it is possible tointroduce one and the same group or else different groups several times.PNA-6 is an example of a multiple conjugation at the carboxy terminusand an additional modification at the amino terminus. The fluorosceinand the amino linker were firstly synthesized successively at thecarboxy terminus. After the PNA moiety had been synthesized, ahydroxyethylglycine-t building block was coupled on, in the last cycle,with this building block being reacted with C16-phosphorylating reagent7. PNA-1 and PNA-2 are compounds of Formula I which are only modifiedwith a phosphoryl radical at the carboxy terminus (q=0). This substanceclass is likewise novel and part of the subject matter of the invention.

FIGS. 5 a and 5 b show some examples of compound types for theN-terminal modification of the compounds of Formula I. Compound type Ais obtained by reacting the terminal hydroxyl group of the PNA with thephosphorylation reagent 1. Compound type B is obtained by reacting theterminal amino group of the PNA with the biotin-phosphoramidite 5.Compound type C is obtained by successively reacting the PNA having aterminal hydroxyl group with the spacer-18 phosphoramidite 9, aminomodifier-5 phosphoramidite 12 and lexitropsin. Compound type D isobtained by successively reacting the PNA having a terminal hydroxylgroup with the spacer-9 phosphoramidite 8 and the cyanine-3phosphoramidite 10. Compound type E is obtained by successively reactingthe PNA having a terminal hydroxyl group with the bifunctionalfluorescein-phosphoramidite 4, the spacer-9 phosphoramidite 8, and theC16-phosphorylating reagent 7. The steps which additionally have to becarried out, such as oxidation and protecting group elimination, aredescribed in the examples.

An example of a carboxy-terminal modification of PNA obtained using aphosphoramidite of the Formula V D is depicted in FIG. 7. In this case,the starting material is a bishydroxyethylsulfone support I (FIG. 6),which, after the Dmt group has been eliminated with 3% trichloroaceticacid, is reacted with the phosphoramidite of the Formula V D usingtetrazole as catalyst. After oxidizing with iodine water, theamino-terminal Mmt group is eliminated with 3% trichloroacetic acid andthe PNA moiety is then synthesized using methods known from theliterature, for example using the Mmt method which is explained below.An alternative method for the carboxy-terminal modification uses CPGsupports which are modified in accordance with the radical to beintroduced, and consequently contain the fluorescein radical, forexample (FIG. 8). This method will be explained using the example of aPNA derivative which is modified amino-terminally with a hexadecylphosphate radical and carboxy terminally with a fluorescein phosphate.The fluorescein support 3 (FIG. 6) is first of all detritylated withtrichloroacetic acid and then condensed with the amino modifier C6phosphoramidite 13 (FIG. 4 d) using tetrazole. After oxidizing withiodine water and eliminating the Mmt group, the PNA moiety can besynthesized using customary methods. In the last cycle, ahydroxyethylglycine-based PNA building block (Formula V A, u′=2,V′=oxygen) is coupled on, with this building block being reacted asshown in FIG. 9 after eliminating the Dmt protecting group using the C16phosphorylating reagent 7. The doubly modified PNA derivative isobtained after eliminating all the protecting groups and cleaving fromthe CPG support.

In embodiments, the invention provides a process for preparing a PNAderivative of Formula I in which q is 0. In these embodiments, theprocess comprises linking the C-terminus of an amidonucleic acid, whichis optionally N-terminally protected, to a solid phase-boundphosphorylating reagent, or binding an amidonucleic acid which isphosphorylated C-terminally to a solid support. Optionally, the backboneof the PNA oligomer is then extended by sequentially coupling withamidonucleic acid monomers. Optionally, the N-terminus of the PNAoligomer is then deprotected. In embodiments, the PNA is prepared usingt-butyloxycarbonyl (BOC), 9-fluorenylmethoxycarbonyl (Fmoc), ormonomethoxytrityl (Mmt) protecting groups.

In embodiments, the invention provides a process for preparing a PNAderivative of Formula I in which q is 1, wherein the process comprises:

a) linking the C-terminus of an amidonucleic acid, which is optionallyN-terminally protected, to a solid phase-bound phosphorylating reagent,or binding an amidonucleic acid which is phosphorylated C-terminally toa solid support,

b) optionally, extending the backbone of the PNA oligomer bysequentially coupling with amidonucleic acid monomers,

c) optionally, deprotecting the N-terminally protected PNA backbone,

d) coupling a phosphorus (III) or a phosphorus (IV) group to theN-terminus of the PNA backbone using activated phosphorylating reagentsoptionally containing a spacer,

e) optionally, repeating step d), and

f) optionally, oxidizing the phosphorus (III) group to a phosphorus (V)group.

EXAMPLES

The following examples are presented to more fully describe selectedembodiments of the invention. The following examples are not intended,and should not be construed, to limit the invention in any way.

The preparation of the following compounds is described by way ofexample:

wherein the sequences of the 13 bases are in each case described by SEQID NO:53, and z″ in each case is 10:

SEQ ID NO:53 5′-T A T T C C G T C A T-3′ (PNA-1 to PNA-7)

Example 1 Synthesizing the PNA Chain

The following reagents were used for preparing the PNA moiety:

-   -   1. Phosphoramidite reagent (0.1 M in acetonitrile (ACN))    -   2. Mmt-PNA monomers and/or Dmt-oeg-PNA monomers (0.2 M in        DMF:ACN (1:1; v:v))    -   3. Anhydrous ACN (≦30 ppm of water)    -   4. Trichloroacetic acid (3%) in dichloromethane (DCM)    -   5. Acetic anhydride, 2,6-lutidine in THF (1:1:8; v:v:v); (Cap A)    -   6. N-Methylimidazole (16%) in THF; (Cap B)    -   7. Iodine solution (0.05 M) in THF, water, pyridine (7:2:1;        v:v:v)    -   8. Washing solution (THF, water, pyridine (7:2:1; v:v:v))    -   9. Tetrazole (0.3 M) in ACN    -   10. HBTU; 0.2 M in DMF:ACN (1:1; v:v)    -   11. DIPEA; 0.2 M in DMF:ACN (1:1; v:v)    -   12. DMF (>99.5%)    -   13. Solid phase support: aminopropyl-CPG (550 Å) loaded with        Mmt-aminohex-1-yl hemisuccinate (for PNA-hexylamides).

The Mmt/acyl-protected or Dmt/acyl-protected oeg monomers were preparedas has already been described (Breipohl et al. (1997) Tetrahedron 53,14671-14686). The loading of aminopropyl-CPG with the Mmt-aminohex-1-ylhemisuccinate has likewise already been described (Will et al. (1995)Tetrahedron 51, 12069-12082). The derivatized CPG supports arecommercially available (Glen Research Corporation, Sterling, Va. 20164,U.S.A.). The PNA syntheses were in general carried out on a scale offrom 2 to 5 μmol.

The following cycle was used for the PNA synthesis:

-   -   1. Step of washing with ACN    -   2. Deprotecting the Mmt group or the Dmt group by treating with        3% trichloroacetic acid (TCA) in DCM; 110 sec.    -   3. Step of washing with DMF/ACN (1:1)    -   4. Neutralizing with DIPEA in DMF/ACN (1:1)    -   5. Coupling on the monomeric building block by preactivating (15        min) with HBTU/DIPEA/PNA monomer (ratio 1:1:1; total volume 450        μl)        -   charging the solid phase and coupling (45 min)    -   6. Step of washing with ACN    -   7. Capping with acetic anhydride/N-methylimidazole    -   8. Step of washing with ACN    -   9. New cycle

Example 2 Synthesizing acetyl-tat tcc gtc at-aminohexyl-p (PNA-1)

The Dmt protecting group was first of all eliminated from thebishydroxyethylsulfonyl support 1 (1 μmol, FIG. 6) by treating with 3%trichloroacetic acid. The free hydroxyl function was then reacted withthe amino modifier C6 phosphoramidite 13 (FIG. 4 d) using tetrazole ascatalyst. The reaction employs an excess of the phosphorylating reagent13 (approx. 25-fold), as an 0.3 M solution inacetonitrile/tetrahydrofuran (1:1; v:v), and the tetrazole (approx.50-fold; 0.5 M in acetonitrile). After the condensation took place,oxidation was effected using an iodine solution (0.05 M intetrahydrofuran/water, pyridine (7:2:1; v:v:v)). After that, the PNAmoiety was prepared by solid phase synthesis as described in Example 1.In the last cycle, the free amino function was acetylated by treatingwith the capping reagent. This prevented the PNA from being degradedamino-terminally during deprotection with conc. ammonia. Finally, thePNA was cleaved from the support, and the protecting groups were removedat the same time, by treating with conc. ammonia at 50° C. overnight.103 OD (260 nm) of the desired crude product was obtained. The crudeproduct was purified by preparative polyacrylamide (PAA) gelelectrophoresis. The desired product band was eluted with 0.2 Mtriethylammonium bicarbonate buffer and desalted through a Bond-Elute C18 column (1 g). 23.3 OD was obtained. The product was analyzed bynegative ion mass spectrometry, which confirmed the calculated mass(calc. 3166.2; found 3166.8).

Example 3 Synthesizing acetyl-tat tcc gtc at(eo)-p (PNA-2)

The preparation was effected, in a 1 μmol synthesis, in an analogousmanner to that described in Example 2. After the Dmt protecting groupwas eliminated from the support (FIG. 6), the free hydroxyl function wasreacted with the phosphoramidite of Formula V D using tetrazole ascatalyst. The reaction employs an excess of the phosphoramidite (approx.20-fold), as a 0.1 M solution in acetonitrile/tetrahydrofuran (1:1; v:v)and the tetrazole (approx. 50-fold; 0.5 M in acetonitrile). After thecondensation took place, oxidation was effected using an iodine solution(0.05 M in tetrahydrofuran/water, pyridine (7:2:1; v:v:v)). 50 OD ofcrude product was obtained after cleaving with ammonia. 45 OD of thiscrude product was purified by electrophoresis through a 15% PAA gel.13.2 OD of product, having a molecular weight of 3052.9 (calc. 3052.9),was obtained.

Example 4 Synthesizing aminohexyl-p-t(oeg) at tcc gtc at-aminohexyl-p(PNA-3)

The preparation was effected, in a 1 μmol synthesis, in an analogousmanner to that described in Example 2. However, after the carboxyterminus and the PNA moiety had been synthesized, ahydroxyethylglycine-based building block having thiamine as thenucleobase (oegT) was coupled on in the last cycle. After the Dmt groupwas eliminated, the free hydroxyl function was coupled to the aminomodifier C6 phosphoramidite 13 (FIG. 4 d) using tetrazole as catalystand subsequently oxidized with iodine water. The oligomer was cleavedfrom the support, and all the base-labile protecting groups were removedat the same time, by treating with conc. ammonia at 50° C. The terminalMmt protecting group was then removed by treating with 80% acetic acid.130 OD of the crude product was obtained, with this group product beingpurified by gel electrophoresis. 22.5 OD of product, having a molecularweight of 3303.8 (calc. 3305.0), was obtained.

Example 5 Synthesizing biotin-p-t(oeg) at tcc gtc at-aminohexyl-p(PNA-4)

The preparation was effected, in a 0.5 μmol synthesis, in an analogousmanner to that described in Example 2. However, after synthesizing thecarboxy terminus and the PNA moiety, a hydroxyethylglycine-basedbuilding block having thiamine as the nucleobase (oegT) was coupled onin the last cycle. After eliminating the Dmt group, the free hydroxylfunction was coupled to the biotin phosphoramidite 5 (FIG. 4 b) usingtetrazole as catalyst and subsequently oxidized with iodine water anddetritylated with trichloroacetic acid. The oligomer was cleaved fromthe support, and all the protecting groups were removed at the sametime, by treating with conc. ammonia at 50° C. 37 OD of the crudeproduct was obtained, with this crude product being purified by gelelectrophoresis. 22.5 OD was obtained.

Example 6 Synthesizing p-t(oeg) at tcc gtc at-aminohexyl-p-fluorescein(PNA-5)

The synthesis was effected in analogy with Example 2 proceeding from thefluorescein-support 3 (FIGS. 6 a and 8). The Dmt protecting group waseliminated from the fluorescein-support 3 by treating with 3%trichloroacetic acid. The free hydroxyl function was then reacted withthe amino modifier C6 phosphoramidite 13 (4 d) using tetrazole ascatalyst. After condensation had taken place, oxidation was effectedusing an iodine solution (0.05 M in tetrahydrofuran/water, pyridine(7:2:1; v:v:v)). After that, the PNA moiety was prepared by solid phasesynthesis as described in Example 1. A hydroxyethylglycine-basedbuilding block having thiamine as nucleobase ((t)oeg) was coupled on inthe last cycle. After eliminating the Dmt group, the free hydroxylfunction was coupled to the phosphorylating reagent 1 (FIG. 4 a) usingtetrazole as catalyst and subsequently oxidized with iodine water.Finally, the PNA was cleaved from the support, and the protecting groupswere removed at the same time, by treating with conc. ammonia at 50° C.overnight. 61 OD (260) of the crude product was obtained, with thiscrude product being purified by preparative polyacrylamide (PAA) gelelectrophoresis. The desired product band was eluted with 0.2Mtriethylammonium bicarbonate buffer and desalted through a Bond-Elut C18column (1 g). 5.6 OD was obtained. The product was analyzed by negativeion mass spectroscopy, which showed the calculated mass (calc. 3709.5;found 3706.3).

Example 7 Synthesizing C16-p-t(oeg) at tcc gtcat-aminohexyl-p-fluorescein (PNA-6)

The synthesis was effected in analogy with Example 6 starting from 1μmol of fluorescein support 3 (FIGS. 6 a and 8). Ahydroxyethylglycine-based building block having thiamine as thenucleobase ((t)oeg) was coupled on in the last cycle. However, aftereliminating the Dmt group, the free hydroxyl function was coupled to theC16 phosphorylating reagent 7 (FIG. 4 c) using tetrazole as catalyst andsubsequently oxidized with iodine water. Finally, the PNA was eliminatedfrom the support, and the protecting groups were removed at the sametime, by treating with conc. ammonia at 50° C. overnight. 61 OD (260) ofthe desired crude product was obtained, with this crude product beingpurified by preparative polyacrylamide (PAA) gel electrophoresis. Thedesired product band was eluted with 0.2M triethylammonium bicarbonatebuffer and desalted through a Bond-Elut C18 column (1 g). 4.6 OD wasobtained. The product was analyzed by negative ion mass spectrometry,which showed the calculated mass (calc. 3934, found 3931).

Example 8 Determining the Melting Temperatures

The melting temperatures were determined using an HP 8452A diode-arrayspectrophotometer, an HP 89090A Peltier element and HP TemperatureControl Software Rev. B5.1 (from Hewlett Packard). Measurements weretaken in 0.5° C./min steps in 140 mM KCl, 10 mM sodium dihydrogenphosphate, 0.1 mM EDTA (pH 7.4) as the buffer. The oligomerconcentration was from 0.5 to 1 OD₂₆₀ per ml.

Surprisingly, the doubly phosphoryl-modified PNA-5 and PNA-6 derivativeshaving two or three negative charges exhibited an equally good or betterdegree of binding towards complementary DNA and RNA than did theuncharged PNA (reference substance).

PNA derivative T_(m) (DNA) T_(m) (RNA) Reference Ac-HN-tat tcc gtcat-hex 41.9° C. 56.6° C. PNA-5 p-t(oeg) at tcc gtc at-aminohexyl-p-41.8° C. 56.9° C. fluorescein PNA-6 C16-p-t(oeg) at tcc gtc at- 44.1° C.56.9° C. aminohexyl-p-fluorescein

Example 9 Determining Cell Uptake after Fluorescence Labeling

COS cells were allowed to grow to confluence in Dulbecco's MEM, whichwas supplemented with 10% FCS, in 5 cm Petri dishes. The cells werewashed twice with serum-free DMEM. An area of approx. 1 cm² wasscratched out in the middle of the Petri dish using a sterile needle.The PNA solution (10 μM) under investigation was applied in this area.The dish was incubated at 37° C. under a CO₂ atmosphere. After 2, 4 and16 hours, the cells were examined by fluorescence microscopy. For this,the cells were washed four times with serum-free DMEM, covered with acover slip, and evaluated under the fluorescence microscope or by phasecontrast. PNA-5 and PNA-6 were examined by fluorescence microscopy.

In this connection, it was found that the hexadecyl-PNA derivative(PNA-6) was taken up more efficiently into the cells than the PNA withno hexadecyl radical.

Example 10 Inhibiting Cell Proliferation with PNA-6

The sequence of PNA-6 is directed against the translation start of theHa-ras mRNA. REH cells (human pre-B leukemia cells, DSM ACC 22) or A549tumor cells were cultured, at 37° C. and under 5% CO₂, in OptiMEM (GibcoBRL) containing 10% fetal calf serum (FCS, GIBCO-BRL). The cell densityfor the assay was approx. 1×10⁶/ml. The PNA-6 (10 μM) was incubated withthe cells in 24-well plates. After incubating at 37° C. and under 5% CO₂for 96 hours, the cell density was determined. Mean values for the celldensity were determined from 3 individual wells at a given PNAconcentration. It was found that PNA-13 inhibits proliferation of theREH cells. After >4 days of incubation, the inhibition brought about byPNA-6 was greater than that brought about by a correspondingphosphorothioate oligonucleotide.

Example 11 Synthesizing aminohexyl-p-spacer18-p-t(oeg) at tcc gtcat-aminohexyl-p (PNA-7)

The synthesis was effected in a 1 μmol synthesis, in an analogous mannerto that described in Example 2. However, after the carboxy terminus andthe PNA moiety had been synthesized, a hydroxyethylglycine-basedbuilding block having thymine as the nucleobase (oegT) was coupled on inthe last cycle. After eliminating the Dmt group, the free hydroxylfunction was coupled to the spacer 18 phosphoramidite (FIG. 4 c) and,after detritylating once again, to the amino modifier C6 phosphoramidite13 (FIG. 4 d) using tetrazole as catalyst and subsequently oxidized withiodine water. The oligomer was cleaved from the support, and all thebase-labile protecting groups were removed at the same time, by treatingwith conc. ammonia at 50° C. The terminal Mmt protecting group was thenremoved by treating with 80% acetic acid. 57 OD of the crude product wasobtained, with this crude product being purified by gel electrophoresis.7.4 OD of product, which exhibits the expected molecular weight of3647.5 (calc. 3648.5) in the mass spectrum, was obtained.

LIST OF ABBREVIATIONS

-   -   ACN Acetonitrile    -   BOC tert-butyloxycarbonyl    -   C, c pseudo-iso-cytosine    -   COS CV1 origin SV 40    -   CPG controlled pore glass    -   DCM Dichloromethane    -   DIPEA Diisopropylethylamine    -   DMEM Dulbecco's MEM    -   DMF Dimethylformamide    -   Dmt Dimethoxytrityl    -   DNA deoxyribonucleic acid    -   DNP Dinitroaryl    -   FITC fluorescein isothiocyanate    -   Fmoc Fluorenylmethoxycarbonyl    -   HATU O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium        hexafluorophosphate    -   HBTU O-(benzotriazol-1-yl)-1,1,3,3-tetramethyluronium        hexafluorophosphate    -   Hex —NH—(CH₂)₆—OH    -   MEM modified Eagle's minimal essential medium    -   Mmt Monomethoxytrityl    -   OD optical density    -   Oeg N-(2-hydroxyethyl)glycine    -   PAA Polyacrylamide    -   PG protecting group    -   PNA polyamide nucleic acid    -   RNA ribonucleic acid    -   TBTU O-(benzotriazol-1-yl)-1,1,3,3-tetramethyluronium        tetrafluoroborate    -   TCA trichloroacetic acid    -   THF Tetrahydrofuran    -   TR acid-labile protecting group

All references cited herein are hereby incorporated in their entirety byreference.

1. A process for preparing a PNA derivative of Formula I

wherein q is 0; D′ is, independently of each other, hydroxyl, mercapto,amino, alkylamino, or acylamino; V is oxygen, sulfur, or NR₁; V′ is,independently of any other V′, oxygen, sulfur, NR₁,U—(CR₃R₄)_(u′)—C(O)—NH, or U—(CH₂CH₂O)_(u′)—CH₂—C(O)—NH; U is,independently of any other U, oxygen, sulfur, or NH; u′ is,independently of any other u′, from 1 to 10; W and W′ are, independentlyof each other, oxygen, sulfur, or NR₁; Y and Y′ are, independently ofeach other, hydroxyl, mercapto, oxyanion, thioate, or NR₁R₂; X and X′are, independently of each other, U—(C₂-C₂₂-alkanediyl)—U,U—(CH₂CH₂—O)_(u′), a labeling group, a group for crosslinking, a groupwhich promotes intracellular uptake, or a group which increases thebinding affinity of the PNA derivative for nucleic acids; Z and Z′ are,independently of each other, hydroxyl, mercapto, oxyanion, thioate,NR₁R₂, C₁-C₂₂-alkyl, C₁-C₈-arylalkyl, C₁-C₂₂-alkyl-U, C₁-C₈-arylalkyl-U,hydroxy-C₁-C₁₈—U, aminoalkyl-U, mercaptoalkyl-U, a group of the formulaR₇(CH₂CH₂—O)_(m′), wherein R₇ is hydroxyl, amino, or C₁-C₂₂-alkoxy, andm′ is from 1 to 100, a labeling group, a crosslinking group, a groupwhich promotes intracellular uptake, or a group which increases thebinding affinity of the PNA derivative for nucleic acids; R₁ and R₂ are,independently of each other, a radical consisting of hydrogen orC₁-C₆-alkyl, preferably hydrogen, R₃ and R₄ are, independently of eachother, a radical consisting of hydrogen or C₁-C₆-alkyl, or the radicalof an amino acid side chain, wherein adjacent radicals R₃ and R₄ in V′can also form a C₅-C₈-cycloalkyl ring; n is from 0 to 10; m is from 0 to10; and wherein {POLY} is described by Formula II

wherein {BLOCK} is, independently of any other {BLOCK}, a group selectedfrom Formula IIIA,

Formula IIIB,

and Formulae IV A to IV G,

wherein each {BLOCK} building block can be different, and wherein z″ isfrom 0 to 100; G is (CR₅R₆)_(u′), C(O)NH—(CR₁R₂)_(t′), orC(O)NH—(CH₂CH₂O)_(u′)—CH₂CH₂, wherein t′ is from 2 to 10; A is,independently of any other A, a group (CR₁R₂)_(s), wherein s is from 1to 3; B is, independently of any other B, either an aromatic radical, aheteroaromatic radical, hydrogen, hydroxyl, or C₁-C₁₈-alkyl, or anucleobase which occurs naturally, and is customary in nucleotidechemistry, or which does not occur naturally, or its prodrug form; D is,independently of any other D, a group (CR₃R₄)_(t), wherein t is from 2to 10; E is, independently of any other E, a group (CR₅R₆)_(u′), R₅ andR₆ are, independently of each other, a radical consisting of hydrogen,C₁-C₆-alkyl, or the radical of an amino acid side chain, whereinadjacent R₅ and R₆ radicals can form a C₅-C₈-cycloalkyl ring or a spirocompound; wherein R₁, R₂, R₃, R₄, and u′ are as defined above; andphysiologically tolerated salts of the PNA derivative of Formula I, withthe provisos that at least one Y, Y′, Z, or Z′ radical is hydroxyl,mercapto, oxyanion, or thioate, and that at least one B radical is anucleobase; said process comprising a) linking the C-terminus of anamidonucleic acid, which is optionally N-terminally protected, to asolid phase-bound phosphorylating reagent, or binding an amidonucleicacid which is phosphorylated C-terminally to a solid support, b)optionally, extending the backbone of the PNA oligomer by sequentiallycoupling with amidonucleic acid monomers, and c) optionally,deprotecting the N-terminus of the PNA oligomer.
 2. The process asclaimed in claim 1, wherein the PNA is prepared using t-butyloxycarbonyl(BOC), 9-fluorenylmethoxycarbonyl (Fmoc), or monomethoxytrityl (Mmt)protecting groups.
 3. The process as claimed in claim 1, wherein the PNAis prepared using solid supports.
 4. The process as claimed in claim 3,wherein CPG, tentagel, or aminomethylpolystyrene is used as the solidsupport.
 5. The process for preparing a PNA derivative of the Formula Ias claimed in claim 1, further comprising purifying the PNA derivativeusing chromatography or electrophoresis.
 6. The process as claimed inclaim 5, wherein the PNA derivative is purified using chromatographyusing a basic stationary phase and a gradient of an acid orsalt-containing eluent.
 7. The process as claimed in claim 6, whereinthe stationary phase is an anion exchanger or a mixed-mode phase.
 8. Aprocess for preparing a PNA derivative of Formula I

wherein q is 1; D′ is, independently of each other, hydroxyl, mercapto,amino, alkylamino, or acylamino; V is oxygen, sulfur, or NR₁; V′ is,independently of any other V′, oxygen, sulfur, NR₁,U—(CR₃R₄)_(u′)—C(O)—NH, or U—(CH₂CH₂O)_(u′)—CH₂—C(O)—NH U is,independently of any other U, oxygen, sulfur, or NH; u′ is,independently of any other u′, from 1 to 10; W and W′ are, independentlyof each other, oxygen, sulfur, or NR₁; Y and Y′ are, independently ofeach other, hydroxyl, mercapto, oxyanion, thioate, or NR₁R₂; X and X′are, independently of each other, U—(C₂-C₂₂-alkanediyl)—U,U—(CH₂CH₂—O)_(u′), a labeling group, a group for crosslinking, a groupwhich promotes intracellular uptake, or a group which increases thebinding affinity of the PNA derivative for nucleic acids; Z and Z′ are,independently of each other, hydroxyl, mercapto, oxyanion, thioate,NR₁R₂, C₁-C₂₂-alkyl, C₁-C₈-arylalkyl, C₁-C₂₂-alkyl-U, C₁-C₈-arylalkyl-U,hydroxy-C₁-C₁₈—U, aminoalkyl-U, mercaptoalkyl-U, a group of the formulaR₇(CH₂CH₂—O)_(m′), wherein R₇ is hydroxyl, amino, or C₁-C₂₂-alkoxy, andm′ is from 1 to 100, a labeling group, a crosslinking group, a groupwhich promotes intracellular uptake, or a group which increases thebinding affinity of the PNA derivative for nucleic acids; R₁ and R₂ are,independently of each other, a radical consisting of hydrogen orC₁-C₆-alkyl, preferably hydrogen, R₃ and R₄ are, independently of eachother, a radical consisting of hydrogen or C₁-C₆-alkyl, or the radicalof an amino acid side chain, wherein adjacent radicals R₃ and R₄ in V′can also form a C₅-C₈-cycloalkyl ring; n is from 0 to 10; m is from 0 to10; and wherein {POLY} is described by Formula II

wherein {BLOCK} is, independently of any other {BLOCK}, a group selectedfrom Formula IIIA,

Formula IIIB,

and Formulae IV A to IV G,

wherein each {BLOCK} building block can be different, and wherein z″ isfrom 0 to 100; G is (CR₅R₆)_(u′), C(O)NH—(CR₁R₂)_(t′), orC(O)NH—(CH₂CH₂O)_(u′)—CH₂CH₂, wherein t′ is from 2 to 10; A is,independently of any other A, a group (CR₁R₂)_(s), wherein s is from 1to 3; B is, independently of any other B, either an aromatic radical, aheteroaromatic radical, hydrogen, hydroxyl, or C₁-C₁₈-alkyl, or anucleobase which occurs naturally, and is customary in nucleotidechemistry, or which does not occur naturally, or its prodrug form; D is,independently of any other D, a group (CR₃R₄)_(t), wherein t is from 2to 10; E is, independently of any other E, a group (CR₅R₆)_(u′), R₅ andR₆ are, independently of each other, a radical consisting of hydrogen,C₁-C₆-alkyl, or the radical of an amino acid side chain, whereinadjacent R₅ and R₆ radicals can form a C₅-C₈-cycloalkyl ring or a spirocompound; wherein R₁, R₂, R₃, R₄, and u′ are as defined above; andphysiologically tolerated salts of the PNA derivative of Formula I, withthe provisos that at least one Y, Y′, Z, or Z′ radical is hydroxyl,mercapto, oxyanion, or thioate, and that at least one B radical is anucleobase; said process comprising a) linking the C-terminus of anamidonucleic acid, which is optionally N-terminally protected, to asolid phase-bound phosphorylating reagent, or binding an amidonucleicacid which is phosphorylated C-terminally to a solid support, b)optionally, extending the backbone of the PNA oligomer by sequentiallycoupling with amidonucleic acid monomers, c) optionally, deprotectingthe N-terminally protected PNA backbone, d) coupling a phosphorus (III)or a phosphorus (IV) group to the N-terminus of the PNA backbone usingactivated phosphorylating reagents optionally containing a spacer, e)optionally, repeating step d), and f) optionally, oxidizing thephosphorus (III) group to a phosphorus (V) group.